Influenza accounts for approximately 200,000 hospitalizations and 36,000 deaths in the United States each year. The elderly, children under age five, individuals with underlying health problems, and pregnant women are typically at increased risk for severe disease.
Three types of influenza (A, B, and C) are associated with human disease. Influenza C is not considered clinically important, while influenza B is generally self-limiting. In contrast, influenza A can cause severe, often life-threatening disease, and is of significant medical importance. Type A influenza is further divided into subtypes based on two viral surface proteins, hemagglutinin (HA) and neuraminidase (NA), with 16 known HA and 10 NA subtypes. HA and NA influence viral host range and not all HA or NA subtypes are capable of promoting human infection. Human infection is usually limited to viruses with the NA subtypes N1 or N2. Seasonal influenza in humans is predominantly due to HA subtypes H1, H2, and H3, while human disease acquired from avian sources can include H5, H7, and H9. Highly pathogenic avian influenza types H5N1 and H7N9 are of recent concern.
Influenza is highly adept at escaping detection by the immune system. Antigenic variation occurs by two distinct mechanisms. Antigenic drift results as mutations accumulate in the genes for HA and NA, leading to variants that are no longer recognized by antibodies raised against the original form. In contrast, antigenic shift occurs when two different influenza viruses infect the same cell and shuffle their HA or NA genes, creating new combinations. For example, a cell infected with an avian H5N1 virus and an H3N2 virus could generate new H5N2 or H3N1 recombinants. The entire human population is usually extremely susceptible to new recombinants, and antigenic shift can therefore trigger a global pandemic. Three pandemics occurred in the twentieth century: 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2). The 1918 pandemic was devastating and is estimated to have caused the death of over 21 million people. The pandemic strain of 2009, A(H1N1)pdm09, originally called the “swine flu,” set off the first global pandemic of the twenty-first century. The extensive antigenic variation of influenza means the influenza vaccine must be reformulated each year using the strains currently in circulation and presents challenges to developing antibody-based diagnostic assays.
Rapid diagnostic tests could greatly reduce the disease burden due to influenza. Since viral shedding occurs before symptoms appear, rapid diagnostic tests for influenza could identify infected individuals before they spread the disease. Isolation of infected individuals has been shown to be especially valuable in settings with a high concentration of highly vulnerable individuals, such as pediatric hospitals and nursing homes. In addition, rapid diagnostics for influenza would improve treatment. In the absence of a diagnosis, patients may be given antibiotics which are only effective against bacterial infections. In addition, antiviral drugs for influenza are only effective if given within 48 hours of onset of symptoms and resistance to antivirals can develop rapidly. The neuraminidase inhibitors oseltamivir (Tamiflu®) and zanamivir (Relenza®) prevent the action of NA, which promotes viral release from infected cells. Recently, resistance to the neuraminidase inhibitors has developed. During the 2008-2009 influenza season the seasonal H1N1 strain was resistant to oseltamivir, while the seasonal H3N2 variant and the novel, pandemic variant of H1N1 were susceptible to oseltamivir. This situation confounds the ability to generate empirical treatment recommendations.
There are two current approaches for influenza diagnosis. The first approach, molecular polymerase chain reaction (PCR)-based methods, detects viral nucleic acids and can provide rapid (2-4 hours) and sensitive identification of viruses. However, PCR tests are expensive and require well-trained personnel, perishable reagents, and sophisticated equipment, making them unsuitable as rapid point-of-care diagnostics. Further, constant change in the viral genome results in the need for constant development and validation of nucleic acid primers. PCR can be used to identify strains with resistance to antiviral drugs, but only after the resistant isolates have been identified and sequenced and primers recognizing the mutations conferring resistance have been developed and validated.
The second approach for influenza diagnosis includes rapid antigen detection assays developed to diagnose seasonal influenza from biological samples. These point-of-care tests can return results in 15 minutes, but have certain limitations. Some commercially available tests detect only influenza A, some detect both influenza A and B but do not distinguish between them, and others detect and distinguish between influenza A and B. At present, commercially available rapid diagnostic tests do not differentiate between seasonal influenza A and highly pathogenic avian influenza. Rapid diagnostic tests are generally highly specific (>90%), but not very sensitive (20%-70%). As a result, the Centers for Disease Control and Prevention (CDC) has advised that negative test results should not be used to make treatment or infection-control decisions, particularly when influenza virus is circulating in a community. Antibody-based rapid tests may fail to detect new isolates resulting from antigenic shift. Further, antibody-based tests may need to be reengineered for new H and N viruses.
A need exists for the development of rapid diagnostic tests that detect influenza in a patient clinical sample regardless of HA or NA subtype, distinguish influenza from other pathogens in order to minimize false positives, and determine susceptibility of influenza subtypes to NA inhibitors such as oseltamivir and zanamivir. Such improved tests would identify infection with sensitive or resistant strains, direct treatment of individuals infected with susceptible isolates, avoid over-use of neuraminidase inhibitors in treating resistant strains of influenza, and minimize inappropriate use of antibiotics, which are ineffective for viral infections.